Methods for improving feed utilization and lactation in ruminant animals

ABSTRACT

Disclosed are methods for improving feed utilization efficiency of ruminents and for improving lactation of lactating ruminants. The methods involve administering certain tetraamide compounds such as 2,2&#39;,2&#34;, 2&#39;&#34;,-1,2-ethanediyldinitrilo) tetrakis-[N,N-bis(2-methylpropyl)acetamide].

FIELD OF THE INVENTION

The invention relates to methods for improving feed utilization and lactation in ruminant animals. In particular, the invention relates to methods of improving ruminant feed utilization and lactation by administering one or more tetraamides.

BACKGROUND OF THE INVENTION

It is well established that improvements in feed utilization efficiency in ruminant animals can be achieved by altering the fermentation process which takes place in the rumen. Ruminant animals utilize their food by degrading the carbohydrates contained therein to pyruvate and metabolizing the pyruvate to volatile fatty acids (VFAs) such as acetate, propionate and butyrate. These VFAs are absorbed from the gut and are employed for energy production which can then be channeled into growth, lactation, etc., by the ruminant.

The process of formation of acetate in the rumen is one of the major inefficiencies in the digestive process. Since acetate is made by the degradation of a pyruvate molecule, each molecule of acetate which is produced is accompanied by a one carbon molecule which subsequently results in the formation of methane. Most of the methane produced is lost through eructation. Since butyrate is made from two molecules of acetate, each molecule of butyrate involves the loss to the animal of two molecules of methane, with all of the associated energy.

Thus, the efficiency of carbohydrate utilization (carbohydrates being the major nutritive portion of ruminant animals' feed) can be increased by treatments which encourage the animal to produce propionate rather than acetate or butyrate from carbohydrates. Further, the efficiency of feed use can be effectively monitored by observing the production and concentration of propionate in the rumen. If the animal is making more propionate, it will be found to be using its feed more efficienctly. A reduction in the amount of methane produced has also been observed to be a good indication of increased feed efficiency.

In this regard, compounds which increase the amount of propionate produced by a ruminant animal enhance the efficiency of feed utilization and have been observed to provide many beneficial results. For instance, it has been disclosed that administration of certain antibiotics which promote production of propionate can be used to promote growth rates of ruminant animals (see, e.g., Celmer et. al., U.S. Pat. No. 4,431,801; Maehr, U.S. Pat. No. 4,218,560). Additionally, propionate-increasing substances have also been helpful in improving milk production in lactating ruminants (e.g., Scheifinger, U.S. Pat. Nos. 4,430,328 and 4,336,250). It is thus highly desirable to develop compounds which can increase the production of propionate in ruminant animals such as cattle, sheep or goats in order to improve feed utilization by the animals which will promote the growth of the animals and also achieve improved lactation as well.

SUMMARY OF THE INVENTION

The present invention is directed to a method for increasing the efficiency of feed utilization by ruminant animals comprising administering to said ruminant animals an effective amount of one or more tetraamide compound of the formula: ##STR1## wherein each R, independently, represents a straight or branched chain alkyl of one to six carbon atoms, or the physiologically acceptable salts thereof.

The present invention is also directed to a method for improving lactation in lactating ruminant animals comprising administering to said animals a lactation improving amount of one or more of the compounds of Formula I.

Compositions comprising one or more compounds of Formula I in combination with an inert carrier are also within the scope of the invention.

The compound of Formula I wherein each R group is iso-butyl is most preferred for use in the methods and compositions of the invention. Said compound is novel and is, therefore, also within the scope of the present invention. Preferably in the compound of Formula I, R will be a group having three to five carbon atoms, such as tert-butyl, n-butyl, isopropyl or isobutyl. Most preferably, R will be isobutyl.

As used herein, the term "effective amount" refers to that amount of one or more compounds of the invention, when administered to a ruminant animal, that is sufficient to increase the growth rate and/or feed conversion efficiency of the treated ruminant animals without resulting in any significant adverse side effect; the term "lactation improving amount" referes to that amount of one or more compounds of the invention, when administered to a lactating ruminant, that is sufficient to cause an observable improvement in milk production without resulting in any significant adverse side effects.

DETAILED DESCRIPTION OF THE INVENTION

The tetramide compounds of Formula I can be prepared in two steps. The first step involves forming a chloroamide compound of the formula: ##STR2## wherein R has the same meaning as previously defined, by reacting chloroacetyl chloride with an appropriate amine compound of the formula:

    HN(R).sub.2                                                (III)

wherein R has the same meaning as previously defined.

The reaction is carried out in a suitable solvent such as diethyl ether for a time and at a temperature sufficient to form the desired chloroamide compound of Formula II. A typical temperature is between about -10° C. and room temperature and a typical time is about one hour. The second step involves forming the desired compound of Formula I by reacting an appropriate chloroamide compound of Formula II with ethylenediamine in the presence of a suitable base such as diisopropylethylamine, diisobutylamine, triethylamine, or the like in a suitable solvent medium such as dimethoxyethane. The reaction is allowed to proceed, typically under an inert gas such as argon or nitrogen, at a sufficient temperature for a sufficient time such that the desired compound of Formula I is formed. A typical temperature is about reflux and a typical time is about 16-24 hours.

The salts of the compounds useful in the methods of this invention are physiologically acceptable salts derived from physiologically acceptable acids. Such physiologically acceptable acids include inorganic acids, e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, and the like, as well as organic acids, such as aliphatic mono- or dicarboxylic acids, phenyl-substituted alkanecarboxylic acids, hydroxyalkanecarboxylic acids or alkanedicarboxylic acids, aromatic acids, aliphatic or aromatic sulfonic acids, and the like.

Physiologically acceptable salts of these acids include therefore, for example, the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and the like.

The salts of the tetraamide of the present invention can be prepared according to procedures commonly employed for the preparation of anionic salts. For example, the free base form of a tetraamide compound is dissolved in a suitable solvent, and an aqueous or organic solution of the desired acid is added to the tetraamide solution. The tetraamide anionic salts can be isolated by filtration and recrystallization or by evaporation of the solvent and purification.

In the present invention, an effective amount of tetraamide compound is administered to a ruminant animal in order to improve the efficiency of feed utilization by the animal. It is well established that the administration of substances which improve feed utilization efficiency through increases in propionate production also acts to improve growth rates in ruminant animals. The improved growth rates result from the additional energy made available by the more efficient processing of feed. Therefore the feed utilization method of the invention also encompasses growth promotion activity.

It has been further established that propionate-increasing compounds which improve feed utilization efficiency can also be effective in increasing milk production in lactating ruminant animals. In the lactating ruminant, energy for lactation is most often the most limiting factor in milk production, and improvements in feed utilization efficiency often provide the energy needed for increased lactation. However, improving feed utilization by increasing the animal's production of propionate will not always ensure that the milk produced by the lactating ruminant will be of sufficient quality. In milk production, acetate is required in order to synthesize milk fat, while propionate is utilized to produce glucose, and has very little effect on the milk fat produced. In addition, butyrate is degraded into acetate units and then it too can be used in long chain fatty acid synthesis producing the milk fat.

Accordingly, in order to increase milk production in lactating ruminants it is necessary to increase production of propionate, but not at the expense of significant decreases in acetate or butyrate production. Significant reductions in acetate and butyrate production result in a milk product of reduced milk fat content, rendering the milk less economically desirable in the United States at the present time. It is desirable, therefore, that a compound desirable for use in promoting lactation be one that improves propionate production without significantly lowering acetate and butyrate levels.

The tetraamide compounds of the present invention provide a significant increase in propionate levels without causing a significant decrease in acetate or butyrate levels, and thus can be used to promote lactation in lactating ruminants. In fact, an increase in acetate level is sometime observed. A method of improving lactation in ruminants is thereby provided which comprises administering to lactating ruminant animals a lactation improving amount of one more tetraamide compounds of the present invention. The administration of the tetraamide(s) may be direct, or a composition for promoting lactation may be prepared which comprises a lactation-promoting amount of the tetraamide of Formula I and an inert carrier.

Although it is not desired to be bound by any particular mechanism or theory, the tetraamide compounds of the present invention are believed to function by selectively inhibiting the growth of a number of species of bacteria which are commonly found in the rumen of ruminant animals. When included in a growth medium designed to support the growth of pure rumen bacterial species, one or more tetraamides inhibit growth of microorganisms such as Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Streptococcus bovis, and the like. In contrast, organisms such as Selenomonas ruminantium usually are only slightly affected. In general, the tetraamide compounds of the present invention seem more active against gram-positive bacteria than gram-negative bacteria.

The tetraamide compounds of the present invention can be administered to increase feed utilization efficiency of ruminants in any way which ensures that the animals receive an effective amount of one or more of the desired compounds. It is preferred that one or more compounds be administered orally and at an effective amount of from about 0.2 to about 100 milligrams (mg) per kilogram (kg) of ruminant body weight per day. It is particularly preferred that the effective amount is between about 1 to about 10 mg/kg of body weight per day of one or more tetraamide compounds of the present invention.

The tetraamide compounds of the present invention can be administered to improve lactation of lactating ruminants in any way which ensures that the animals receive a lactation improving amount of one or more of the desired compounds. A lactation improving amount is typically that amount that results in an increase in the volume of milk produced by about 2 to about 15 percent relative to untreated animals. It is preferred that one or more compounds be administered orally and at a lactation improving amount of from about 0.2 to about 100 mg per kg of ruminant body weight per day. It is particularly preferred that the lactation improving amount is between about 1 and about 10 mg/kg of body weight per day of one or more tetraamide compounds of the invention.

The exact effective amount and/or lactation improving amount of one or more tetraamides to be employed will vary depending upon factors such as species of animal, or the size, weight, age and health of the animal. In particular cases, the concentration can be determined by conventional dose titration techniques.

The most practical way to administer the tetraamide compound of the present invention is to add an effective amount (or lactation improving amount) of one or more tetraamides into the animal's feed. This can be done by directly adding a desired amount of the tetraamide(s) to the feed supply, or by first forming the compound(s) into a premix composition for subsequent addition to the feed. Such a composition preferably comprises one or more tetraamide compounds of Formula I above, along with an inert carrier, the amount of tetraamide(s) being sufficient to result in increased feed utilization and/or improved lactation in an appropriate ruminant animal upon administration to said animal. Suitable carriers include ground corn, barley, soybean meal, wheat, soy flour, or any similar low-priced, edible material. It is desired that the amount of one or more tetraamide compounds used in the composition be sufficient to comprise from about 5 to about 1000 parts per million in the animal feed. It is particularly preferred that the tetraamide comprises from about 30 to about 300 parts per million of the ultimate animal feed.

The form of the additive to the feed is not crucial, and alternative forms for administering the tetraamide compound may be employed. For instance, the compound can be incorporated into tablets, drenches, salt blocks, paste, boluses, or capsules and doped to the animals. Formulation of the compounds in such dosage forms can be accomplished by means of methods well known in the veterinary pharmaceutical art. Each individual dosage unit should contain a quantity of the compound which has a direct relation to the proper daily dose for the animal to be treated, as discussed above.

The following examples are provided in order to further illustrate the present invention and should not be construed as limiting the invention in any way.

EXAMPLE 1 Preparation of Chloroacetic Acid, N,N-diisobutylamide

To a suspension of diisobutylamine (110 g, 149 mL), sodium carbonate (80 g) and ether (400 mL) at -10° C. was added chloroacetyl chloride (85 g, 0.75 mol). The reaction mixture was maintained below room temperature during the addition. The mixture was stirred for 45 minutes after the addition was complete. The solid was removed by filtration, and the filtrate was concentrated resulting in a residue. The residue was dissolved in methylene chloride, extracted with 1N HCl followed by dilute sodium carbonate and dried over solid sodium sulfate which resulted in an aqueous and organic layer. Concentration of the organic layer afforded 80.8 g of a light yellow liquid. The NMR spectrum of this light yellow liquid was consistent with the desired product and indicated that the compound was >95% pure.

EXAMPLE 2 Preparation of 2,2',2",2'"-(1,2-ethane-diyldinitrilo)tetrakis[N,N-bis(2-methyl-propyl)acetamide]

A solution of ethylenediamine (0.3 g, 0.005 mol), triethylamine (0.05 mol), dimethoxyethane (20 mL) and chloroamide 6 (5.13 g, 0.025 mol) was heated to reflux under nitrogen for 24 hours. The dimethoxyethane was removed from the crude reaction by rotary evaporator which left a residue. The residue was partitioned between 1N HCl and methylene chloride. The bulk of the product was in the organic layer. The original organic layer was concentrated to give a solid product which was recrystallized from heptane. The yield was 2.6 g of a white solid. The field desorption mass spectrum (FDMS), nuclear magnetic resonance (NMR) spectrum and the infrared spectrum of the white solid supported the structure of the desired product.

EXAMPLE 3 Effect of Tetraamide Compound on Rumen Bacterial Growth

The compound of Example 2 above was placed in a growth culture medium of five rumen bacterial species, and growth of the treated cultures was monitored and compared with growth in control media. The results are presented in Table 1 below. The tetraamide compound of the present invention substantially prevented the growth of R. flavefaciens and B. fibrisolvens. In addition, the growth of S. bovis and B. ruminicola was inhibited. The growth of S. ruminantium was almost unafffected by the tetraamide. In general, the tetraamide appeared to be more active against gram-positive bacteria than gram-negative bacteria.

                  TABLE 1                                                          ______________________________________                                         RESPONSES OF BACTERIA TO                                                       TREATMENT WITH TETRAAMIDE                                                      BACTERIA          OD.sub.660.sup.1                                                                        SD.sup.2                                                                              RESPONSE.sup.3                               ______________________________________                                         Ruminococcus                                                                             control     1.131    0.033                                                                               0                                          flavefaciens                                                                             w/tetraamide                                                                               -0.026   0.030                                                                               101                                        Butyrivibrio                                                                             control     1.813    0.073                                                                               0                                          fibrisolvens                                                                             w/tetraamide                                                                               -0.058   0.003                                                                               103                                        Streptococcus                                                                            control     1.694    0.051                                                                               0                                          bovis     w/tetraamide                                                                               1.141    0.100                                                                               33                                         Bacteroides                                                                              control     1.648    0.011                                                                               0                                          ruminicola                                                                               w/tetraamide                                                                               1.228    0.283                                                                               25                                         Selenomonas                                                                              control     1.704    0.049                                                                               0                                          ruminantium                                                                              w/tetraamide                                                                               1.616    0.068                                                                               5                                          ______________________________________                                          .sup.1 Each value is the mean of triplicate samples.                           .sup.2 SD is the standard deviation.                                           .sup.3 Responses represent inhibition of growth as a percent of the            controls and are calculated as follows: Response = 100 × [1 -            (experimental optical density at 660 nanometers (OD.sub.660)/control           OD.sub.660)                                                              

EXAMPLE 4 Fermentation Experiments

Rumen bacterial suspensions were prepared using rumen fluid obtained from a cannulated steer. The rumen fluid was mixed in a Waring blender under CO₂ for one minute and then strained through four layers of cheesecloth. The strained fluid was then diluted with three volumes of suitable growth medium to obtain final bacterial suspensions. Various levels of the tetraamide (of Example 2) from 1 mg/mL solutions or suspensions in methanol were added to 50-mL vials. The methanol was evaporated by drying in a 39° C. oven for 24 hours, and 0.4 g of chopped, dried alfalfa (sieved, 1 mm mesh) was added to each vial. The final bacterial suspensions obtained as described above were added to the vials, 20 mL of suspension per vial, and the suspensions were incubated for 20 hours with an aerobic CO₂ atmosphere, shaking at 39° C.

The suspensions were then processed for determination of volatile fatty acids (VFA). The VFAs were determined by the following procedure. After filtration of the VFA sample through 0.2 micron filters to remove particulate matter, samples were transferred to 2-mL vials. The VFA concentrations of the samples were determined by GLC, using a Hewlett Packard (HP) 5880 gas chromatograph on a 2-meter glass column packed with 0.3% carbowax 20 M on Carbopack C. Injector and detector temperatures were set at 200° C. while the oven temperature was programmed from 85° C. to 140° C. at a 4° C./minute. Carrier flow rate was set at 25 mL per minute. The results of the VFA determinations are presented in Table 2.

The results indicated that the tetraamide compound was responsible for an increase in the level of propionate in the rumen bacterial suspension, and this increase was greater with a larger dosage of the tetraamide. In addition, there was an increase in acetate level and a slight decrease in butyrate level.

                  TABLE 2                                                          ______________________________________                                         VFA CONCENTRATIONS IN                                                          TREATED BACTERIAL SUSPENSIONS                                                  Concentrations of VFAs (millimoles per liter)                                  SD = Standard Deviation                                                        Micro-                                                                         grams                                                                          of                                                                             Tetra-                                                                         amide Ace-          Prop-                 Total                                per mL                                                                               tic    SD     ionic SD   Butyric                                                                              SD   VFA   SD                             ______________________________________                                         0     64.0   6.1    46.0  3.5  23.6  1.9  133.6 9.6                            2.5   70.0   6.8    51.2  4.7  26.4  2.2  147.6 11.4                           5     66.1   4.1    47.7  5.4  26.4  2.2  147.6 11.4                           10    57.3   2.8    44.6  3.7  21.5  0.9  123.4 6.5                            20    70.2   3.0    52.0  1.6  24.2  0.6  146.4 4.3                            40    78.6   4.1    60.9  2.5  16.2  0.7  155.7 6.8                            ______________________________________                                     

I claim:
 1. A method for increasing the efficiency of feed utilization by ruminant animals comprising administering to said animals an effective amount of a compound of the formula: ##STR3## wherein each R, independently, represents a straight or branched chalk alkyl of one to six carbon atoms, or the physiologically acceptable salts thereof.
 2. The method of claim 1 wherein each R independently represents an alkyl group of three to five carbon atoms.
 3. The method of claim 1 wherein each R independently represents tert-butyl, iso-butyl, n-butyl or iso-propyl.
 4. The method of claim 1 wherein R is iso-butyl.
 5. The method of claim 1 wherein said growth promoting amount is between about 0.2 and 100 mg per kg of body weight per day.
 6. The method of claim 1 wherein said growth promoting amount is between about 1 and 10 mg per kg of body weight per day. 